EP1583970B1 - Elisa method for the detection of guanylate binding protein 1 (gbp-1) - Google Patents

Abstract

A method is disclosed for the identification and/or quantification of GBP-1 or of fragments of said protein in the culture residue of a tissue sample, a body fluid sample or a sample from a cell culture residue.

Description

The present invention relates to methods of identifying and / or quantifying GBP-1 or fragments of this protein in the culture supernatant of a tissue sample, a sample of body fluid or a sample of cell culture supernatant.

The endothelium is a key organ in numerous physiological and pathophysiological processes such as cell-directed immune response, menstruation, wound healing, inflammation, allergy, cardiovascular disease and tumor growth. Endothelial pathophysiology is inextricably linked to the activation of endothelial cells.

Activation of the endothelium is a complex process controlled by a variety of soluble factors that circulate in the blood or are released by neighboring cells ( Fig. 1A ). As a result of this process, the physiology and morphology of the endothelial cells are adapted to the respective requirements in the tissue. The focus is on the control of cell proliferation, apoptosis, invasion, migration and leukocyte adhesion of endothelial cells, which regulate vascular newbend and return vasculature and leukocyte extravasation ( Fig. 1A ).

The large number of factors involved suggests that several factors can control the same phenotype and can be grouped into equivalent groups ( Fig. 1B ). For example, the angiogenic growth factors activate basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF). endothelial cell proliferation, whereas the inflammatory cytokines interleukin (IL) -1α, IL-1β, tumor necrosis factor (TNF) -α and interferon (IFN) -γ inhibit proliferation and increase leukocyte adhesion of endothelial cells.

Currently, there are no suitable methods for determining where and when the various factors in inflammatory tissues act on the endothelial cells. Therefore, little is known about the spatial and temporal distribution of the various activation states of endothelial cells in the context of inflammatory diseases.

The development of inflammatory responses and consequent inflammatory diseases is a very complex sequence (cascade) of different and synergistic effects of inflammatory factors such as cytokines, which make it difficult to analyze a defined stage of inflammatory responses and / or to reliably predict their further development , Due to this complexity of the ongoing reactions one speaks in this context of so-called cytokine network.

Initial work to identify a molecular marker that indicates activation of endothelial cells by the above-mentioned inflammatory cytokines in tissue has led to comparative studies of gene expression in cultured endothelial cells in the presence of different activation conditions. With this approach it was possible to isolate a gene whose expression in endothelial cells is selectively induced by inflammatory cytokines ( Fig. 2A ) (Guenzi et al., 2001; Lubeseder-Martellato et al., 2002). This gene encodes guanylate binding protein-1 (GBP-1), which belongs to the large GTPase protein family.

To determine whether GBP-1, as in cultured cells, also indicates inflammatory cytokine activation in vascular endothelial cells in human tissues, immunohistochemistry studies were performed to detect GBP-1 using specific monoclonal antibodies. In addition were histological sections of healthy skin and of skin diseases with an inflammatory component, such as psoriasis, drug reaction and Kaposi's sarcoma ( Fig. 2B ). Common to all the skin diseases mentioned is that focal lesions contain numerous inflammatory cells which release the same inflammatory cytokines, which also lead to increased GBP-1 expression. In the vessels of healthy skin, GBP-1 could not be detected in any case. In contrast, in each inflammatory disease studied, individual vessels were clearly positive for GBP-1 ( Fig. 2B , Arrows). These results showed that GBP-1 actually displays inflammatory activation of endothelial cells also in human tissues and can be used as a molecular marker for the detection of this activation in tissues (Lubeseder-Martellato et al., 2002, Guenzi et al., 2001). However, this use as a molecular marker was limited to solid tissue samples because the results described demonstrated that the GBP-1 protein is a cell-acting protein located in the cytoplasm of the cell. Corresponding evidence included, for example, the recovery of solid tissue samples from patients. However, removal of solid tissue samples from inflammatory tissue is associated with adverse effects for patients and is difficult.

Induction of inflammatory cytokine GBP-1 expression is associated with inhibition of cell proliferation in endothelial cells. Therefore, it was investigated whether GBP-1 mediates inflammatory cytokine-induced proliferation inhibition. For this endothelial cells were transduced with retroviral vectors, which cause the constitutive expression of GBP-1 (GBP-1 vector) or an antisense -GBP-1 RNA (AS vector) ( Fig. 3A ). Western blot analyzes revealed that endothelial cells transduced with the GBP-1 vector expressed GBP-1 very strongly ( Fig. 3B ). In cells transduced with the AS vector, the induction of GBP-1 expression by IL-1β was efficiently blocked (Fig. Fig. 3B ). Subsequent proliferation experiments with the different transduced cell cultures showed that GBP-1 actually inhibits angiogenic growth factor-induced cell proliferation ( Fig. 3C , white bars) and, in addition, that inflammatory cytokines are necessary for the proliferation of Endothelial cells can inhibit ( Fig. 3D ). The latter is expressed in the fact that in cell cultures expressing antisense -GBP-1 RNA the inhibitory effect of inflammatory cytokines on cell proliferation is markedly reduced ( Fig. 3D , black bars) (see Guenzi et al., 2001).
In addition, GBP-1 inhibits the expression of matrix metalloproteinase-1 and, concomitantly, the invasion of endothelial cells (see Guenzi et al., 2003).

Further studies on the structural / functional relationship of GBP-1 showed that intriguingly, the adhesion of endothelial cells to leukocytes, which is also induced by inflammatory cytokines, is not affected by GBP-1 (Guenzi et al., 2001). Thus, GBP-1 is a novel molecular marker for inflammatory vascular activation that selectively controls the antiproliferative effect of inflammatory cytokines on endothelial cells.

So far it has been shown that GBP-1 is selectively induced by inflammatory cytokines and that this process correlates with an anti-angiogenic effect on the cells in question (Lubeseder-Martellato et al., 2002 and Guenzi et al., 2001). While the induction of GBP-1 over its antiproliferative activity could in principle be exploited for medical purposes in the context of anti-angiogenic therapy, the use of inflammatory cytokines for such purposes is indiscussable due to the pleiotropic and thus adverse effects of these cytokines.

Although there is a need for suitable molecular markers for inflammatory diseases, a variety of cytokines and factors involved in the development and development of inflammatory diseases are described in the prior art as being unsuitable for this purpose because of their instability. In addition, a quantification of individual inflammatory cytokines or factors is not sufficient for a clear finding and therefore requires the determination of a variety of different cytokines and factors that show their respective effect only in a so-called "cytokine network".

The object of the present invention was thus to provide a method which allows a simple and targeted analysis of the expression of GBP-1. These procedures should provide information about the stage and progression of an inflammatory response in an individual or in an in vitro model without the need for elaborate analysis and quantitation of many different inflammatory factors of the so-called cytokine network.

This object is achieved by providing the embodiments characterized in the claims.

1. (a) In Kontakt bringen der Probe mit einem ersten Rezeptor, der GBP-1 spezifisch bindet; und
2. (b) Nachweis einer spezifischen Bindung des Rezeptors mit GBP-1 oder einem Fragmente dieses Proteins.

Accordingly, the present invention relates to an in vitro method as defined in the claims for identifying and / or quantifying GBP-1 or fragments of this protein in the culture supernatant of a tissue sample, a sample of body fluid or a sample of cell culture supernatant, the method comprising the following steps :

1. (a) contacting the sample with a first receptor that specifically binds GBP-1; and
2. (b) Detecting specific binding of the receptor with GBP-1 or a fragment of this protein.

The term fragment of GBP-1 describes fragments of the protein having the biological activity of GBP-1 as described in the prior art as well as in this application. According to the invention, the Bagriff fragments of the protein which are formed by cleavage, eg enzymatic cleavage, and are indicative of inflammatory diseases.
The term body fluids in connection with the invention encompasses all types of body fluids, possibly diluted or concentrated. Examples are blood / serum, plasma, amniotic fluid, cerebrospinal fluid, cerebrospinal fluid, cerebrospinal fluid, sputum, pharynx and pharyngeal secretions and other mucosal secretions, synovial fluid, ascites, tears, lymph fluid and urine.
The term "specific binding" according to the invention describes a specific interaction or interaction between a receptor and a ligand. Such a ligand is GBP-1 or fragments of this protein. The specific interaction or interaction can be characterized by a "key-lock" principle. The receptor and ligand have structures or motifs that specifically match one another, such as an antigenic determinant (epitope) that interacts with the antigen-binding site of an antibody. Accordingly, specific interaction is in contrast to a more universal, nonspecific interaction.
It has been shown that GBP-1 is a marker protein for inflammatory reactions, which surprisingly is secreted by, among others, endothelial cells and monocytes. This surprising finding makes it possible to analyze GBP-1 in culture supernatant of tissue samples, samples of body fluid or samples of cell culture supernatants and to make a statement about the stage of an inflammatory disease. Secreted GBP-1 can be detected easily and quickly with the method according to the invention and thus serves as a disease-associated diagnostic parameter.
The detection of inflammatory activation of endothelial cells and monocytes in the body fluid of patients with the method according to the invention is on the basis of the surprising finding of particular importance in inflammatory diseases, bacterial and viral infectious diseases (AIDS, Menigitis), allergies, transplantation reactions, cardiovascular and tumor diseases etc. In addition, these are of importance for determining the response in patients receiving treatment with inflammatory cytokines (eg interferon-α); please refer FIG. 5 ,
The detection, or the quantified amount of GBP-1 in a sample of a body fluid of a patient allows conclusions about the degree of activation of endothelial cells and monocytes and thus a statement about the clinical picture of the patient.
Methods for obtaining said samples are known to those skilled in the art. Optionally, the process according to the invention additionally comprises one or more washing steps before or after each process step. These washing steps serve to minimize unspecific reactions (false positive or false negative detection) and may improve the sensitivity of the process. Suitable wash buffers and their composition are known in the art in principle; see eg Harlow and Lane. Preferred are physiological buffer solutions.

• (a') Markieren der in der Probe enthaltenen Proteine; oder
• (a") Markieren des ersten Rezeptors.

Die in der Probe enthaltenen Proteine und/oder der erste Rezeptor können beispielsweise chemisch markiert werden, z.B. durch die Kopplung von markierten chemischen Gruppen oder Markern an die freien Aminogruppen von in den Proteinen enthaltenen Cysteine. Beispiele für solche markierte chemische Gruppen sind Gruppen, die spezielle, nachweisbare Radioisotope enthalten. Als Marker können z.B. auch Fluoreszenzfarbstoffe dienen. Ein weiteres Beispiel für entsprechende Marker stellen Nukleinsäuren dar. Die Anwesenheit von auf diese Weise markierten Proteinen oder Rezeptoren in einer Probe kann dann mit geeigneten Primern in einer Polymerasekettenreaktion (PCR) nachgewiesen werden.
Des weiteren ist es möglich Proteine physiologisch zu markieren, d.h. durch den metabolischen Einbau von markierten Molekülen. Zu diesem Zweck werden Zellen beispielsweise mit radioaktiv markierten Metaboliten inkubiert. Proteine, die während dieser Inkubationszeit aus der Biosynthese dieser Zellen hervorgehen und in welche die markierten Metaboliten eingebaut wurden, sind makiert. Dieses Verfahren ist z.B. geeignet, um von antikörperproduzierenden Zellen sezernierte Antikörper zu markieren.A preferred embodiment of the method of the invention further comprises step (a ') or (a ") prior to contacting the first receptor:

• (a ') labeling the proteins contained in the sample; or
• (a ") marking the first receptor.

For example, the proteins and / or the first receptor contained in the sample can be chemically labeled, for example, by the coupling of labeled chemical groups or labels to the free amino groups of cysteines contained in the proteins. Examples of such labeled chemical groups are groups containing specific, detectable radioisotopes. For example, fluorescent dyes can also serve as markers. Another example of corresponding markers are nucleic acids. The presence of proteins or receptors labeled in this way in a sample can then be detected with suitable primers in a polymerase chain reaction (PCR).
Furthermore, it is possible to mark proteins physiologically, ie by the metabolic incorporation of labeled molecules. For this purpose, cells are incubated, for example, with radioactively labeled metabolites. Proteins resulting from the biosynthesis of these cells during this incubation period and into which the labeled metabolites have been incorporated are labeled. This method is useful, for example, to label antibodies secreted by antibody-producing cells.

In another preferred embodiment of the method of the invention, the receptor is immobilized on a surface prior to exposure to GBP-1 or fragments of this protein.
According to an alternative embodiment of the method according to the invention, the receptor is immobilized on a surface after contacting GBP-1 or fragments of this protein.

Receptors can be immobilized in a variety of ways. The corresponding method depends on various factors, such as the type of receptor or the material of the surface. Immobilization can be covalent or by adsorption. According to a preferred embodiment of the method according to the invention, the receptors are proteins, more preferably antibodies. Also preferred is the use of peptides or organic molecules as receptors.
For the immobilization of receptors, which are proteins, methods are described in which they are immobilized directly on a surface by means of passive adsorption. Usually, a corresponding surface consists of a polymer plastic (eg polystyrene, polyvinyl, latex) and used for this purpose in the form of microtiter plates or multi-well plates, membranes or spherical 'beads' (cross-linked polymers in particle form) ( Lowman, Annu. Rev. Biophys. Biomol. Struct. 26 (1997), 401-24 ).

In a further preferred embodiment of the method according to the invention, the material of the surface is selected from a group consisting of sepharose, latex, glass, polystyrene, polyvinyl, nitrocellulose and silicon.
More preferably, the surface in the method according to the invention is a membrane, a bead, a chip or a plate.
Examples of beads are Sepharose beads or latex beads, to which optionally ligands are bound, which promote immobilization of the receptors to the surface. Such ligands are, for example, protein A or protein G, which promote binding of antibody to a surface via the Fc portion of the antibodies. The binding of the receptor to support material can also be achieved by a covalent chemical coupling reaction (eg hydrazide coupling). Example 3 describes a corresponding method. Another example of the immobilization of the receptors to the surface using ligands is the use of biotin and avidin, or streptavidin.
Examples of chips are silicon plates to which a plurality of different or the same receptors can be imobilized in order. This allows the analysis of a variety of different parameters in a sample or the Analysis of a variety of different samples for one or more parameters, eg identification and / or quantitation of GBP-1 or fragments of this protein in different tissue samples, samples of body fluid or samples of cell culture.
Examples of said plates are microtiter plates or multi-well plates. These preferably have 6, 12, 24, 48, 96, 128, 356, 1024 or more wells. Example 4 describes a method in which 96well plates are used.

• (a"') präzzipitieren der Kügelchen mit den daran gebundenen Komplexen aus erstem Rezeptor und GBP-1 oder eines Fragmentes dieses Proteins Kügelchen können aus einer Probe z.B. gravimetrisch präzipitiert werden. Dies kann beispielsweise durch Zentrifugation beschleunigt werden. Entsprechende Verfahren sind dem Fachmann u.a. aus Rehm, Der Experimentator: Proteinbiochemie/Proteomics, Spektrum Akademischer Verlag, 2002 bekannt. Des weiteren wird eine entsprechende Präzipitation in Beispiel 3 beschrieben.

According to a further preferred embodiment of the method, this furthermore comprises the step (a "') before the step of detecting a specific binding:

• (a "') Precipitate the beads with the complexes bound from the first receptor and GBP-1 or a fragment of this protein Beads can be gravimetrically precipitated from a sample, for example, by centrifugation accelerated Rehm, The Experimenter: Protein Biochemistry / Proteomics, Spektrum Akademischer Verlag, 2002. Furthermore, a corresponding precipitation in Example 3 is described.

In a further preferred embodiment of the method according to the invention, the detection of the specific binding in step (b) comprises a gel electrophoretic separation, optionally additionally a Western blot analysis (see Example 3). Corresponding methods are known to the person skilled in the art, inter alia. from Rehm, loc. cit. known.

In a further preferred embodiment of the method of the invention, to detect specific binding of GBP-1 or a fragment of this protein to the first receptor in step (a), the sample is contacted with a second GBP-1 receptor that binds to an epitope of GBP-1 or a fragment of this protein accessible after binding of the first receptor to GBP-1 or a fragment of this protein.

This preferred embodiment relates, for example, to methods which make use of the mechanistic principle of the sandwich ELISA. This principle is well known to those skilled in the art and is i.a. in Stryer, Biochemistry, Spektrum Akademischer Verlag, 1996. A corresponding method is also described in Example 4 attached.

Further, the second receptor for GBP-1 or fragments of this protein is labeled in a preferred embodiment of the method of the invention. Methods which allow labeling of a receptor have been described above and can also be used here.
In addition, it is preferred that the label of the second receptor for GBP-1 or a fragment of this protein comprises a signaling system. Likewise preferred is a specific recognition of the label by a further, third receptor, which comprises a signaling system.
An example of such a signaling system is the above-described isotope tag, which signal is the delivery of radioactive radiation. Likewise, a fluorescent label of the corresponding receptor in the label results in a signal-generating system according to the invention, the signal being the emission of a fluorescence signal after appropriate excitation of the dye.
More preferably, the signaling system according to the invention comprises an enzyme which emits a signal. Examples of such enzymes include alkaline phosphatases, peroxidases, β-galactosidase, glucoamylase, urease, and chloramphenicol acetyltransterase. Corresponding examples and the use of necessary substrates for the detection by means of enzymatic reactions are known to the person skilled in the art, inter alia from the instruction leaflets commercially available detection kits or from Rehm, loc. cit. Such commercially available detection kits often contain antibodies that recognize antibodies of certain species, eg anti-mouse, and to which signaling enzymes are coupled. Thus, corresponding antibodies represent an example of a third receptor recognizing a particular label of the second receptor, namely its Fc portion.

According to the invention, the first and the second receptor and the third receptor are selected from the group consisting of antibodies or defined below thereof and aptamers.
The term peptides usually refers to amino acid chains of up to 30 amino acids. The term polypeptides refers to peptides that usually comprise more than amino acid chains of 30 amino acids and includes proteins.
By the term "low molecular weight substances" or small molecules are meant molecules which are of lower molecular complexity than the macromolecules defined above. In the literature, the term "low molecular weight substances" is not used uniformly. In WO 89/03041 and WO 89/03042 For example, molecules with molecular weights up to 7000 g / mol are described as small molecules. Usually, however, molecular masses between 50 and 3000 g / mol, more often between 75 and 2000 g / mol and usually in the range between 100 and 1000 g / mol. Examples are those skilled in the art from the publications WO86 / 02736 . WO97 / 31269 . US-A-5928868 . US-A-5242902 . US-A-5468651 . US-A-5547853 . US-A-5616562 . US-A-5641690 . US-A-4956303 and US-A-5928643 known. Low molecular weight substances may be organic or inorganic in nature.
The term "antibody" according to the invention comprises polyclonal sera, as well as monoclonal antibodies.
Monoclonal antibodies and methods for their production are known in the art. These are based on a method first described by Köhler and Milstein (1975). This is among others in the laboratory manual by Harlow and Lane (Antibodies, A laboratory manual; Cold Spring Harbor Laboratory; (1988); Chapter 6 ) described in detail. By definition are also included bispecific antibodies, synthetic antibodies and fragments of these antibodies. These are the fragments Fab, Fv or scFv and chemically modified derivatives of these antibodies or antibody fragments.
Aptamers are known to those skilled in the art in principle from the prior art.
Preferably, the method of the invention is an ELISA, EIA, or RIA.

Corresponding methods are known in the art in principle from Harlow and Lane, loc. cit. and Rehm, loc. cit.

The method according to the invention is preferably carried out automatically. This is i.a. possible through the use of pipetting robots and for an automated evaluation of optimized operations.

Further, the invention relates to the use of body fluid or a sample of cell culture supernatant as defined above for the detection of GBP-1 or fragments of this protein, wherein the positive detection is indicative of the presence of an inflammatory disease.
The other (preferred) embodiments of the use according to the invention correspond to those for the method described above.

• Fig. 1 : Komplexität und Redundanz entzündlicher Endothelzellaktivierung.
  1. (A) Die Aktivierung des Endothels bei entzündlichen Prozessen wird durch eine Vielzahl verschiedener löslicher Faktoren aus dem Blut und von benachbarten Zellen gesteuert. Dabei stehen im Rahmen der Neu- und Rückbildung von Gefäßen, sowie der Extravasation von Leukozyten die Steuerung von Zellproliferation, Apoptose, Invasion, Migration und Adhäsionsfähigkeit für Leukozyten im Vordergrund.
  2. (B) Die Vielzahl der beteiligten Faktoren legt nahe, dass mehrere Faktoren ein- und dieselbe Aktivierung beeinflussen und zu wirkungsgleichen Gruppen zusammengefasst werden können. Gegenwärtig kann nicht bestimmt werden, wann und wo die verschiedenen Faktoren auf die einzelnen Endothelzellen einwirken. Darüber hinaus sind die Beziehungen der meisten Aktivierungsarten zueinander weitgehend unbekannt. Es ist zu bestimmen, ob alle Aktivierungen gleichzeitig in einer Zelle auftreten können (I) oder aufgrund zellbiologischer Restriktionen zeitlich, beziehungsweise räumlich separiert sein müssen (II).
• Fig.2 : Expression von GBP-1 in kultivierten Endothelzellen und in entzündlichen Erkrankungen der Haut.
  1. (A) Western Blot-Analyse der GBP-1 Expression in Endothelzellen, die mit den aufgeführten Faktoren für 24 h stimuliert worden waren. Folgende Konzentrationen wurden eingesetzt: IFN-γ (100 U/ml), IL-1α (5 ng/ml), IL-1β (200 U/ml), TNF-α (300 U/ml), IL-4 (10 U/ml), IL-6 (50 U/ml), IL-10 (50 ng/ml), IL-18 (100 ng/ml), Oncostatin M (10 ng/ml), MCP-1 (50 ng/ml), PF4 (25 ng/ml), SDF-1α (200 ng/ml), bFGF (10 ng/ml), VEGF (10 ng/ml), Ang-2 (800 ng/ml) und PDGF B/B (100 ng/ml). Der gleichzeitige Nachweis des Zytoskelettproteins Aktin zeigt, dass gleiche Proteinmengen aufgetragen wurden.
  2. (B) Induktion der GBP-1-Expression in vaskulären Endothelzellen bei Hauterkrankungen mit einer inflammatorischen Komponente. Indirekte Immunfluoreszenzfärbung von GBP-1 (grün) und dem Endothelzell-assoziierten Antigen CD31 (rot) in Gewebeschnitten von gesunder Haut, Kaposi-Sarkom, entzündlicher Arzneimittelgegenreaktion der Haut und Psoriasis. Die Überlagerung der Bilder zeigt eine Ko-Expression (gelb) von GBP-1 und CD31 (weiße Pfeile). (Modifiziert nach (Lubeseder-Martellato, Guenzi et al. 2002))
• Fig.3 : GBP-1 vermittelt die antiproliferative Wirkung inflammatorischer Zytokine in Endothelzellen.
  1. (A) Schematische Darstellung des retroviralen Expressionsvektors pBabePuro (Kontrollvektor, K-Vektor) in den die cDNA von GBP-1 in beiden Orientierungen für die konstitutive Expression von GBP-1 (GBP-1-Vektor) und für die Expression einer GBP-1-antisense-RNA (AS-Vektor) eingesetzt wurde.
  2. (B) GBP-1-Expression in K-, GBP-1-, und AS-Vektor transduzierten Endothelzellen, die entweder unbehandelt waren oder über einen Zeitraum von 24 h mit 20 U/ml IL-1β stimuliert wurden. Der Nachweis der GBP-1-Expression erfolgte mittels WesternBlot-Analyse mit einem polyklonalen anti-GBP-1-Antikörper. Die gleichzeitige Färbung mit Aktin zeigt, dass gleiche Proteinmengen aufgetragen wurden.
  3. (C) Proliferationsexperimente mit K-Vektor- und GBP-1-Vektor-transduzierten Endothelzellen in Anwesenheit steigender Konzentrationen angiogener Wachstumsfaktoren (bFGF und VEGF in Kombination).
  4. (D) Proliferationsexperimente von K-Vektor- und AS-Vektor-transduzierten Endothelzellen in Anwesenheit angiogener Wachstumsfaktoren und aufsteigender Konzentrationen von IL-1β. (Modifiziert nach (Guenzi, Topolt et al. 2001))
• Fig. 4 : Nachweis von GBP-1 Protein in Kulturmedium von IFN-γ stimulierten HUVEC durch ELISA und Immunopräzipitation
  HUVEC wurden Stimuliert mit 100 U/ml IFN-γ (IFN-γ) oder unbehandelt gelassen (Medium). Nach 24 h Kultur wurde das Kulturmedium durch ELISA (A) oder Immunopräzipitation (B) analysiert.
  1. (A) Eine Verdünnungsserie mit rekombiantem aufgereinigtem GBP-1 wurde als Standard verwendet (weiße Säulen). Die Menge von sekretiertem GBP-1 Protein wurde im ELISA bestimmt (graue Säulen). Die Absorption wurde bei 405 nm bestimmt.
  2. (B) Westernblot-Analyse von immunopräzipitierten menschlichem GBP-1 Protein mit monoklonalem anti GBP-1 Antikörper (Klon 1B1) aus dem selben Kulturüberstand wie in (A).
• Fig. 5 : Messung von zirkulierendem GBP-1 im Plasma von IFN-α behandelten Patienten
  Bei Melanom-Patienten, die für 9 bzw. 28 Tage mit IFN-α behandelt wurden, wurde die Konzentration von zirkulierendem GBP-1 im Plasma mittels ELISA bestimmt. Dargestellt ist der Anstieg der GBP-1 Konzetration von Tag 9 zum Tag 28 bei drei Pateinten. ELISA-Mikrotiterplatten wurden mit einem monoklonalen Ratten anti-GBP-1 Antikörper, Klon 1B1, für 16 h bei 4°C beschichtet. Anschließend wurden die Platten mit PBS-T (0,1 % Tween 20 in PBS) und für 30 min wurden bei Raumtemperatur (RT) freie Bindungsstellen mit PBS-T/BSA 2 % (PBS-TB) abgesättigt. Nach dem absaugen des PBS-TB erfolgte Inkubation (2 h) mit je 100 µl verschiedener Plasma Proben (1:2) bei RT. Als Standard wurde gereinigtes GBP-1-His Protein, verdünnt in Zellkulturmedium (EMB-0,5% FCS) benutzt. Als Negativkontrolle diente BSA. Die Proben wurden 4x mit PBS-T gewaschen, je 100 µl polyklonaler Kaninchen anti-GBP-1 Antikörper (1:500) zugegeben und für 2 h bei RT inkubiert. Nach viermaligen Waschen mit PBS-T erfolgte eine Inkubation (1 h,RT) mit je 100 µl AP-konjugiertem anti-Kaninchen Antikörper (1:500 verdünnt in PBS-TB). Nach vier weiteren Waschschritten wurde der GBP-1 Protein/Antikörper-Komplex durch Inkubation mit 100 µl p-Nitrophenyl Phosphat visualisiert. Die Absorption wurde bei 405 nm im Mikroplatten-Reader gemessen. Die Konzentrationsbestimmung erfolgte anhand einer Standardkurve für die steigende Konzentrationen von gereinigtem GBP-1-His verwendet wurden. Sie Linearität der Messung ist für einen Bereich von 0,1 bis 100 ng/ml gegeben.
• Fig. 6 : Die Sekretion von GBP-1 Protein aus IFN-γ behandelt HUVEC ist nicht zunehmende Zellmembranpermeabilität oder Apoptose zurückzuführen
  Färbung von HUVEC mit dem nicht membrangängigen Farbstoff ,,Dead-Red" (Molekular Probes).
  HUVEC wurden über Nacht in 0,5 % FBS-haltigen EBM-Medium kultiviert und mit 100 U/ml Interferon-γ (IFN-γ, Roche) stimuliert. Kontrollzellen (Medium) wurden unbehandelt belassen. 24 h nach der Stimulierung wurden die Zellen mit Dead-Red Farbstoff angefärbt. Zellen, die eine veränderte Membranpermeabilität aufweisen (schwarze Balken), sind als Prozentsatz der Gesamtzahl (weiße Balken) angegeben.
• Fig. 7 . Der ELISA reagiert spezifisch mit GBP-1 und nicht mit den heterologen Proteinen BSA und eGFP
  1. (A) Die Spezifität des GBP-1-ELISA wurde in mehreren Kontrollexperimenten bestimmt. Zunächst wurde mit dem ELISA eine Verdünnungsreihe von gereinigtem GBP-1-His (ein GBP-1-Protein, das aus Bakterien gereinigt wurde und zum Zwecke der Aufreinigung mit 6 Histidinresten am Carboxyterminus versehen ist) in PBS in den angegebenen Konzentrationen erzeugt und im ELISA gemessen. Hierbei wurde ein konzentrationsabhängiger Anstieg der Absorption bei 405 nm (A₄₀₅) beobachtet (schwarze Balken). Wenn anstelle des anti-GBP-1-Kaninchenserums (wird als zweiter Antikörper zum Nachweis des gebundenen GBP-1 eingesetzt) ein Präimmunserum des Kaninchens verwendet wurde, wurden keine Signale erhalten (weiße Balken). Beim Zusatz aufsteigender Konzentrationen heterologer Proteine [BSA (graue Balken), His-eGFP (gestreifte Balken)] wurden mit dem GBP-1-spezifischen ELISA ebenfalls keine Signale beobachtet.
  2. (B) Der GBP-1-ELISA zeigt eine geringe Reaktivität mit dem GBP-1-homologen GBP-2.
  
  Mit dem GBP-1-ELISA wurden Lösungen untersucht, die aufsteigende Mengen an GBP-1-His (schwarze Balken), His-GBP-1 (weiße Balken, ein GBP-1-Protein, das aus Bakterien gereinigt wurde und zum Zwecke der Aufreinigung mit 6 Histidinresten am Aminoterminus versehen ist) und His-GBP-2 [graue Balken, einem zu GBP-1 homologen Protein (Homologie auf Aminosäureebene 76 %, auf Nukleinsäureebene 82 %) mit 6 Histidinresten am Aminoterminus] enthielten. Der Vergleich der Steigungen der Messwerte für GBP-1-His (schwarze Balken), His-GBP-1 (weiße Balken) und His-GBP-2 (graue Balken) zeigte, dass die Reaktivität dieses ELISA mit GBP-2 im Vergleich zur Reaktivität mit GBP-1 verringert ist und das die Histidinreste am Amino- oder Carboxyterminus die Reaktivität von rekombinantem GBP-1 nicht beeinflussen.
  Zusätzlich wurden immunchemische Blockierungsversuche durchgeführt, um die Spezifität der Bindung von gereinigtem GBP-1-His and die mit Ratten-anti-GBP-1-Antikörpern beschichtete ELISA-Platte zu bestimmen. Hierzu wurde GBP-1-His in verschiedenen Verdünnungen mit (schwarz-weiße Balken) oder ohne (schwarze Balken) Ratten-anti-GBP-1-Antikörper inkubiert und nachfolgend auf die Platte gegeben. In den Ansätzen, in denen GBP-1-His nicht mit Antikörpern vorinkubiert wurde, wurde eine konzentrationsabhängige Erhöhung der Signalstärke beobachtet (schwarze Balken). Wohingegen, durch Vorinkubation mit anti-GBP-1-Antikörpern (schwarz-weiße Balken) die Bindung von GBP-1 an die Plattenoberfläche blockiert werden konnte. In einem vergleichbar durchgeführten Ansatz konnte auch die Bindung von His-GBP-2 durch Vorinkubation mit dem Ratten-anti-GBP-1-Antikörper blockiert werden (grau-weiße Balken).
• Fig. 8 : Bestimmung des linearen Nachweisbereichs des ELISA
  1. (A) Gepoolte Seren gesunder Personen wurden 1:2 (Quadrate), 1:4 (Kreise), 1:8 (Dreiecke) und 1:16 (Rauten) in PBS/2% BSA verdünnt. In die unterschiedlichen Verdünnungen wurden jeweils aufsteigende Konzentrationen von GBP-1-His zugegeben. Nachfolgend wurden alle Proben mit dem GBP-1-ELISA vermessen. Es zeigte sich, dass bei jeder der unterschiedlichen Serumverdünnungen die Nachweissignale (A405) mit der GBP-1-Konzentration zunahmen.
  2. (B) Zwischen 0 und 200 ng/ml wurde bei den in (A) beschriebenen Ansatz eine lineare Zunahme des Signals beobachtet.
• Fig.9 : Bei Patienten mit Entzündungserkrankungen sind erhöhte GBP-1 Konzentrationen im Serum nachweisbar
  Die GBP-1 Konzentration wurde mittels ELISA in gesunden Kontrollpersonen (n = 20) und in Seren von Patienten mit verschiedenen Entzündungserkrankungen (n = 10) gemessen: Systemischer Lupus Erythematosus (SLE) (n = 5) und Arthritis (n = 5), diese Erkrankungen sind generalisierte Entzündungen, und in Patienten mit Erysipel (n=8), eine örtlich begrenzte Entzündung der Haut.
  Die GBP-1 Serumkonzentrationen wurden bestimmt, wobei die Serum-Proben 1:2 verdünnt wurden. Die Konzentration von GBP-1 in den Proben wurde mit Hilfe einer Standardkurve berechnet. Da das ELISA-Verfahren auch GBP-2 erkennt und möglicherweise auch GBP-2 im Serum vorhanden ist, wurden die ermittelte Konzentrationen in relativen Einheiten angegeben.
  Die GBP-1 Serumkonzentrationen waren deutlich erhöht bei den Patienten mit SLE (Median: 46,1 relative Einheiten) und Arthritis (Median: 58,2 relative Einheiten) aber nicht in Patienten mit Erysipel (Median: 8,6 relative Einheiten) im Vergleich zu den Kontrollpersonen (Median: 13,3 relative Einheiten). Die Unterschiede der GBP-1 Konzentrationen bei Patienten mit SLE und Arthritis und der GBP-1 Konzentration in gesunden Kontrollpersonen sind statistisch signifikant (Wilcoxon-Test p < 0,01 für SLE und Arthritis).
• Fig.10 : Bei Patienten mit bakterieller Meningitis sind erhöhte GBP-1-Konzentrationen im Liquor nachweisbar
  In einem Blindversuch wurde mittels ELISA die GBP-1 Konzentration in 17 Liquor-Proben nachgewiesen (Figur 10). Nach Entblindung des Versuches zeigte sich, dass bei Patienten mit bakterieller (Pneumokokkus, Staphylokokkus aureus, Pseudomonas aeruginosa) Meningitis (n = 8) im Vergleich zu gesunden Kontrollpersonen (n = 9) signifikant erhöhte (Wilcoxon-Test p<0,03) GBP-1 Konzentrationen im Liquoren nachweisbar waren (Kontrollpersonen = 36,6 relative Einheiten und bakterieller Meningitis = 105,8 relative Einheiten). Die GBP-1 Liquorkonzentrationen wurden bestimmt wie beschrieben. Die Liquor-Proben wurden 1:2 in PBS verdünnt. Die Konzentration von GBP-1 in der Probe wurde mit Hilfe einer Standardkurve berechnet. Da das ELISA-Verfahren auch GBP-2 erkennt und möglicherweise auch GBP-2 im Liquor vorhanden ist, wurden die ermittelte Konzentrationen in relativen Einheiten angegeben.
  Die GBP-1 Konzentration im Liquor gesundern Kontrollpersonen zeigten keinen statisch signifikanten Unterschied (Wilcoxon-Test p>0,05) zu den GBP-1 Konzentrationen im Serum. Dies weist darauf hin, dass GBP-1 nicht im Liquor gesunder Parsonen angereichert ist und dass der Anstieg der GBP-1 Konzentrationen bei Patienten mit Meningitis krankheitsbedingt ist.

The figures show:

• Fig. 1 : Complexity and redundancy of inflammatory endothelial cell activation.
  1. (A) The activation of the endothelium in inflammatory processes is controlled by a variety of different soluble factors from the blood and from neighboring cells. Within the framework of the new and regression of vessels, as well as the extravasation of leukocytes, the control of cell proliferation, apoptosis, invasion, migration and adhesiveness for leucocytes are in the foreground.
  2. (B) The multiplicity of factors involved suggests that several factors influence one and the same activation and can be grouped into equivalent groups. At present, it is not possible to determine when and where the various factors affect the individual endothelial cells. Moreover, the relationships of most types of activation are largely unknown. It must be determined whether all activations can occur simultaneously in one cell (I) or have to be temporally or spatially separated due to cell biological restrictions (II).
• Fig.2 : Expression of GBP-1 in cultured endothelial cells and in inflammatory diseases of the skin.
  1. (A) Western blot analysis of GBP-1 expression in endothelial cells stimulated with the listed factors for 24 h. The following concentrations were used: IFN-γ (100 U / ml), IL-1α (5 ng / ml), IL-1β (200 U / ml), TNF-α (300 U / ml), IL-4 (10 U / ml), IL-6 (50 U / ml), IL-10 (50 ng / ml), IL-18 (100 ng / ml), Oncostatin M (10 ng / ml), MCP-1 (50 ng / ml), PF4 (25 ng / ml), SDF-1α (200 ng / ml), bFGF (10 ng / ml), VEGF (10 ng / ml), Ang-2 (800 ng / ml) and PDGF B / B (100 ng / ml). Simultaneous detection of the cytoskeletal protein actin shows that equal amounts of protein were applied.
  2. (B) Induction of GBP-1 expression in vascular endothelial cells in skin diseases with an inflammatory component. Indirect immunofluorescence staining of GBP-1 (green) and the endothelial cell-associated antigen CD31 (red) in tissue sections of healthy skin, Kaposi's sarcoma, inflammatory drug-reaction of the skin and psoriasis. The overlay of the images shows a co-expression (yellow) of GBP-1 and CD31 (white arrows). (Modified by (Lubeseder-Martellato, Guenzi et al., 2002))
• Figure 3 : GBP-1 mediates the antiproliferative effect of inflammatory cytokines in endothelial cells.
  1. (A) Schematic representation of the retroviral expression vector pBabePuro (control vector, K vector) into which the GBP-1 cDNA in both orientations for the constitutive expression of GBP-1 (GBP-1 vector) and for the expression of a GBP-1 - antisense RNA (AS vector) was used.
  2. (B) GBP-1 expression in K, GBP-1, and AS vector-transduced endothelial cells either untreated or stimulated with 20 U / ml IL-1β over a period of 24 h. The detection of GBP-1 expression was by Western blot analysis with a polyclonal anti-GBP-1 antibody. The simultaneous staining with actin shows that equal amounts of protein were applied.
  3. (C) Proliferation experiments with K vector and GBP-1 vector transduced endothelial cells in the presence of increasing concentrations of angiogenic growth factors (bFGF and VEGF in combination).
  4. (D) Proliferation experiments of K vector and AS vector transduced endothelial cells in the presence of angiogenic growth factors and increasing concentrations of IL-1β. (Modified according to (Guenzi, Topolt et al., 2001))
• Fig. 4 : Detection of GBP-1 protein in culture medium of IFN-γ stimulated HUVEC by ELISA and immunoprecipitation
  HUVEC were stimulated with 100 U / ml IFN-γ (IFN-γ) or left untreated (medium). After 24 hours of culture, the culture medium was analyzed by ELISA (A) or immunoprecipitation (B).
  1. (A) A serial dilution with recombined purified GBP-1 was used as standard (white columns). The amount of secreted GBP-1 protein was determined by ELISA (gray columns). The absorbance was determined at 405 nm.
  2. (B) Western blot analysis of immunoprecipitated human GBP-1 protein with monoclonal anti-GBP-1 antibody (clone 1B1) from the same culture supernatant as in (A).
• Fig. 5 : Measurement of circulating GBP-1 in the plasma of IFN-α treated patients
  In melanoma patients treated with IFN-α for 9 and 28 days, respectively, the concentration of circulating GBP-1 in the plasma was determined by ELISA. Shown is the increase in the GBP-1 concentration from day 9 to day 28 with three godfathers. ELISA microtiter plates were coated with a monoclonal rat anti-GBP-1 antibody, clone 1B1, for 16 h at 4 ° C. Subsequently, the plates were saturated with PBS-T (0.1% Tween 20 in PBS) and for 30 min at room temperature (RT) free binding sites were saturated with PBS-T / BSA 2% (PBS-TB). After aspirating the PBS-TB, incubation was carried out (2 h) with 100 μl of different plasma samples (1: 2) at RT. As a standard, purified GBP-1-His protein diluted in cell culture medium (EMB-0.5% FCS) was used. The negative control was BSA. The samples were washed 4x with PBS-T, 100 μl polyclonal rabbit anti-GBP-1 antibody (1: 500) added and incubated for 2 h at RT. After washing four times with PBS-T, incubation was carried out (1 h, RT) with 100 μl of AP-conjugated anti-rabbit antibody (diluted 1: 500 in PBS-TB). After four more washes, the GBP-1 protein / antibody complex was visualized by incubation with 100 μl of p-nitrophenyl phosphate. The absorbance was measured at 405 nm in the microplate reader. Concentration was determined using a standard curve for which increasing concentrations of purified GBP-1-His were used. Linearity of measurement is given for a range of 0.1 to 100 ng / ml.
• Fig. 6 : The secretion of GBP-1 protein from IFN-γ treated HUVEC is attributed to non-increasing cell membrane permeability or apoptosis
  Staining of HUVEC with the non-membrane-permeable dye "dead-red" (molecular probes).
  HUVEC were cultured overnight in 0.5% FBS-containing EBM medium and stimulated with 100 U / ml interferon-γ (IFN-γ, Roche). Control cells (medium) were left untreated. 24 hours after stimulation, the cells were stained with Dead-Red dye. Cells exhibiting altered membrane permeability (black bars) are reported as a percentage of the total (white bars).
• Fig. 7 , The ELISA reacts specifically with GBP-1 and not with the heterologous proteins BSA and eGFP
  1. (A) The specificity of the GBP-1 ELISA was determined in several control experiments. First, ELISA was used to generate a dilution series of purified GBP-1-His (a GBP-1 protein purified from bacteria and provided with 6 histidine residues at the carboxy-terminus for purification) in PBS in the concentrations indicated and in ELISA measured. A concentration-dependent increase in absorbance at 405 nm (A ₄₀₅ ) was observed (black bars). When a rabbit preimmune serum was used instead of the anti-GBP-1 rabbit serum (used as the second antibody to detect the bound GBP-1) no signals were obtained (white bars). With the addition of ascending concentrations of heterologous proteins [BSA (gray bars), His-eGFP (striped bars)], no signals were also observed with the GBP-1 specific ELISA.
  2. (B) The GBP-1 ELISA shows low reactivity with the GBP-1 homologous GBP-2.
  
  The GBP-1 ELISA was used to investigate solutions using ascending amounts of GBP-1-His (black bars), His-GBP-1 (white bars, a GBP-1 protein purified from bacteria and for the purpose of Purification with 6 histidine residues at the amino-terminus) and His-GBP-2 [gray bars, a GBP-1 homologous protein (homology at 76% amino acid level, 82% at nucleic acid level) with 6 histidine residues at the amino terminus]. Comparing the slopes of the measured values for GBP-1-His (black bars), His-GBP-1 (white bars) and His-GBP-2 (gray bars) showed that the reactivity of this ELISA with GBP-2 was lower than that of Reactivity with GBP-1 is reduced and that the histidine residues at the amino or carboxy-terminus do not affect the reactivity of recombinant GBP-1.
  In addition, immunochemical blocking assays were performed to determine the specificity of binding of purified GBP-1-His and the rat anti-GBP-1 antibody coated ELISA plate. For this purpose, GBP-1-His was incubated at various dilutions with (black-white bars) or without (black bars) rat anti-GBP-1 antibodies and subsequently added to the plate. In the approaches in which GBP-1-His was not pre-incubated with antibodies, a concentration-dependent increase in signal strength was observed (black bars). Whereas, by pre-incubation with anti-GBP-1 antibodies (black and white bars), the binding of GBP-1 to the plate surface could be blocked. In a similar approach, the binding of His-GBP-2 could also be blocked by preincubation with the rat anti-GBP-1 antibody (gray-white bars).
• Fig. 8 : Determination of the linear detection range of the ELISA
  1. (A) Pooled serums of healthy individuals were diluted 1: 2 (squares), 1: 4 (circles), 1: 8 (triangles), and 1:16 (diamonds) in PBS / 2% BSA. In each case, increasing concentrations of GBP-1-His were added to the different dilutions. Subsequently, all samples were measured with the GBP-1 ELISA. It was found that at each of the different serum dilutions, the detection signals (A405) increased with the GBP-1 concentration.
  2. (B) Between 0 and 200 ng / ml, a linear increase in signal was observed in the approach described in (A).
• Figure 9 : Increased GBP-1 levels in serum are detectable in patients with inflammatory disease
  The GBP-1 concentration was measured by ELISA in healthy controls (n = 20) and in sera from patients with various inflammatory diseases (n = 10): systemic lupus erythematosus (SLE) (n = 5) and arthritis (n = 5), These diseases are generalized inflammation, and in patients with erysipelas (n = 8), a localized inflammation of the skin.
  The GBP-1 serum concentrations were determined with the serum samples diluted 1: 2. The concentration of GBP-1 in the samples was calculated using a standard curve. Since the ELISA method also recognizes GBP-2 and possibly also GBP-2 is present in the serum, the determined concentrations were expressed in relative units.
  The GBP-1 serum concentrations were significantly increased in the patients with SLE (median: 46.1 relative units) and arthritis (median: 58.2 relative units) but not in patients with erysipelas (median: 8.6 relative units) in comparison to the controls (median: 13.3 relative units). The differences in GBP-1 concentrations in patients with SLE and arthritis and GBP-1 concentration in healthy controls are statistically significant (Wilcoxon test p <0.01 for SLE and arthritis).
• Figure 10 : Patients with bacterial meningitis have increased levels of GBP-1 in cerebrospinal fluid
  In a blind test, the GBP-1 concentration in 17 CSF samples was detected by ELISA ( FIG. 10 ). After unblinding the trial, it was shown that in patients with bacterial (pneumococcus, Staphylococcus aureus, Pseudomonas aeruginosa) meningitis (n = 8) significantly increased compared to healthy controls (n = 9) (Wilcoxon test p <0.03) GBP -1 concentrations in the CSF were detectable (controls = 36.6 relative units and bacterial meningitis = 105.8 relative units). The GBP-1 CSF concentrations were determined as described. The CSF samples were diluted 1: 2 in PBS. The concentration of GBP-1 in the sample was calculated using a standard curve. As the ELISA procedure also recognizes GBP-2 and possibly also GBP-2 is present in the CSF, the determined concentrations were expressed in relative units.
  The GBP-1 concentration in cerebrospinal fluid controls showed no statistically significant difference (Wilcoxon test p> 0.05) to the GBP-1 serum concentrations. This indicates that GBP-1 is not enriched in the CSF of healthy parsons and that the increase in GBP-1 concentrations in patients with meningitis is due to disease.

The examples illustrate the invention.

Example 1: Preparation of a vector construct

The 237 bp GBP-1 promoter fragment (pro237-GBP-1) was amplified by PCR amplification (PCR 2 Advantage Kit, Clontech) from the construct pro3757-GBP-1 with the oligonucleotides 5'-ATTTG AAGCTT CTGGTTGAG-3 '[insert a HindIII site (underlined)] or 5'-TGGCTTCTAGCACTTCTG-3 'respectively. The construct pro3757-GBP-1 contains 3757 bp of the 5'-regulatory sequence upstream of the ATG codon of the GSP-1 gene (gi: 4503938, NM_002053) in the vector pT-Adv (Clontech). The 237 bp fragment was ligated in antisense orientation with the vector pT-Adv, cut with HindIII and subcloned into the pGL3-Basic vector (Promega). All constructs were purified with the Endofree Maxi Kit (Qiagen) and sequenced for verification.

Example 2: Establishment of a suitable cell line

HEK 293 T cells (human embryonic kidney epithelial cell line transformed with human adenovirus type 5 (Ad 5) DNA (ATCC CRL 1573), which are additionally transformed with SV40 T antigen) were plated with 3x10 ⁵ cells / well (6-multiwell plate , Corning) seeded 24 hours before transfection. A total of 0.8 μg of plasmid was used per well of the 6-well plate, with the test plasmid pro237-GBP-1 in the ratio 1: 5 with the selection plasmid pBABE-Puro (P. Monini, Laboratory of Virology, Instituto Superiore di Santia, Rome, Italy) was used. The test plasmid pro237-GBP-1 contains the 237 bp promoter fragment coupled to the indicator gene firefly luciferase, the selection plasmid pBABE-Puro the resistance gene puromycin, and is subsequently selected. The transfection of the cells was carried out analogously to the manufacturer's instructions with Effectene (Qiagen) and, after 24 h, selection was started with 0.7 μg / ml puromycin (Sigma). On day 8-10, growth of single clones was observed, which were subsequently trypsinized with cloning rings and transferred to single wells of a 96-well plate (Falcon). At appropriate confluence, the clones were further passaged and assayed for reporter gene activity in the luciferase assay. The clones were stimulated with inflammatory cytokines IFN-γ, IL-1β and TNF-α, and buffer for 5 h and harvested in 1x "passive lysis buffer" (Promega). The lysates were examined for firefly-luciferase activity and correspondingly stable clones were established.

Example 3: Immunoprecipitation of GBP-1

Freshly prepared cell lysates were pre-purified by incubation with 2 μl of non-GBP-1 rabbit preimmune serum and 25 μl of protein A / G agarose beads for at least 3 h at 4 ° C on a paddle plate. After pelletizing the beads, the supernatant was incubated with 25 μl of protein A / G agarose beads and 1 μl of rabbit polyclonal serum against GBP-1 overnight at 4 ° C on a shaking platform. The beads were washed four to five times in PBS. Subsequently, the beads were resuspended in 30 μl Laemmli sample buffer (2X) and boiled for 5 min. The samples were separated on SDS-PAGE (10%) and analyzed in Western blot or autoradiography. For immunoprecipitation of GBP-1 or MMP-1 from cell culture supernatants, 10 ml of culture medium were placed on ice, centrifuged at 1000 rpm for 5 min, filtered through a filter with a pore size of 45 μm and in some cases a pro-nase inhibitor cocktail (0.02%) pancreatic extract, 5 μg / ml pronase, 0.5 μg / ml thermolysin, 3 μg / ml chymotrypsin and 0.33 μm / ml papin). The prepurification was carried out by incubation with 10 μl of rabbit serum and 120 μl of protein A / G agarose beads for more than 3 h at 4 ° C on a shaking platform. After pelletizing the beads, the supernatant was incubated with 120 μl of protein A / G agarose beads and 6 μl of rabbit polyclonal serum against GBP-1 overnight at 4 ° C on a shaking platform. The beads were washed four to five times in PBS. Subsequently, the beads were resuspended in 60 μl PBS + 60 μl Laemmli sample buffer (2X) and boiled for 5 min. Typically, 15 μl of each sample was separated on SDS-PAGE (10%) and analyzed in a Western blot.
An example of the result of detection of GBP-1 by immunoprecipitation from culture medium is in FIG. 4B shown.

Example 4: GBP-1 ELISA

• PBS enthaltend 0, 1 % Tween 20 (PBS-T) und PBS enthaltend 0, 1 % Tween 20 und 2% BSA (PBS-TB).

The following buffers were used for the developed GBP-1 ELISA:

• PBS containing 0.1% Tween 20 (PBS-T) and PBS containing 0.1% Tween 20 and 2% BSA (PBS-TB).

96-well ELISA plates (Nunc-Immuno Plates) were coated with 100 μl / well anti-GBP-1 hybridoma supernatant (diluted 1: 5 with PBS) or with purified receptor in the concentration of 1-5 μg / ml (Incubation for 16 h at 4 ° C). The plates were washed with PBS-T and blocked with PBS-TB for at least 30 minutes at room temperature. The wells were aspirated and incubated as doublets for 2 h at room temperature with 100 μl of the standard (GBP-1-His diluted in cell culture medium containing 5% FBS), the same concentration of BSA as control or with 100 μl of a sample in appropriate dilution (diluted in PBS). The wells were washed four times with PBS-T and incubated with 100 μl of a polyclonal antibody to GBP-1 diluted 1: 500 in PBS-TB for 2 h at room temperature. Subsequently, the wells were washed four times with PBS-T and incubated with 100 μl of an alkaline phosphatase conjugated to an anti-rabbit antibody (Zymed, Berlin, Germany) diluted 1: 500 in PBS-TB for 1 h Room temperature incubated. Subsequently, the wells were washed four times with PBS-T and incubated with 100 μl of p-nitrophenyl phosphate (Zymed). The absorption was measured at 405 nm in a microplate reader. (BioRad). The concentration of GBP-1 in the sample was calculated using the standard curve. The method demonstrated a linearity of 0.1 to 100 ng / ml of GBP-1 / well. The variability of the results in different assays was between 2.3 and 6%.
An example of the result of detection of GBP-1 in culture supernatant by ELISA is in FIG. 4A shown.

The sensitivity of the ELISA was determined from a dilution series of GBP-1-His (a GBP-1 protein purified from bacteria and provided with 6 histidine residues at the carboxy-terminus for purification) in PBS.
The sensitivity of the ELISA was defined as the lowest concentration of GBP-1-His for which the associated reading differed significantly, that is, by at least two standard deviations, from the reading used in the batch without GBP-1-His (plus two standard deviations ). The sensitivity of the ELISA described here was determined to be 12.3 ng / ml.

1. Intra-assay variability.

The reproducibility of the results within one experiment was determined by triplicate measurements. The variability was calculated in the following way: $$\mathrm{variability}\ddot{\mathrm{a}}\mathrm{t}\mathrm{=}\left(\mathrm{standard\; deviation}\mathrm{/}\mathrm{<figure-callout\; id="100"\; label="Average\; X"\; filenames="imgf0003.png,imgf0006.png"\; state="\{\{state\}\}">Average\; </figure-callout>}\right)\mathrm{X}\mathrm{}\mathrm{100}\mathrm{\%}\mathrm{,}$$

• GBP-1-His 400 ng/ml; Intra-assay Variabilität = 2,7 %
• GBP-1-His 180 ng/ml; Intra-assay Variabilität = 2,8 %
• GBP-1-His 40 ng/ml; Intra-assay Variabilität = 2,0 %

Three test solutions with different GBP-1-His concentrations (400 ng / ml, 180 ng / ml, 40 ng / ml) were generated by adding GBP-1-His in serum (diluted 1: 2 in PBS) to a healthy volunteer , Each solution determined the intra-assay variability:

• GBP-1-His 400 ng / ml; Intra-assay variability = 2.7%
• GBP-1-His 180 ng / ml; Intra-assay variability = 2.8%
• GBP-1-His 40 ng / ml; Intra-assay variability = 2.0%

2. Inter-Assay Variability:

To determine the inter-assay variability, the reproducibility of the ELISA was determined in various experiments. For this purpose, each of the solutions described above was measured six times. The inter-assay variability was calculated as described above.
GBP-1-His 400 ng / ml; Inter-assay variability = 4.4%
GBP-1-His 180 ng / ml; Inter-assay variability = 2.8%
GBP-1-His 40 ng / ml; Inter-assay variability = 3.0%

Literature

• Carmeliet. and Jain: Nature 407 (6801): 249-57 (2000 ).
• Folkman: Nat Med 1 (1): 27-31 (1995 ).
• Guenzi, Topolt, Cornali, Lubeseder-Martellato, Jorg, Matzen, Zietz, Kremmer, Nappi, Schwemmle, Hohenadl, Barillari, Tschachler, Monini, Ensoli, and Stürzl: (2001) EMBO J 20 (20): 5568-77 ..
• Guenzi E, Töpolt K, Lubeseder-Martellato C, Jörg A, Naschberger E, Benelli R, Albini A, Stürzl M: (2003) The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP 1 expression. EMBO J. 22 (15): 3772-82
• Harlow and Lane, "Antibodies, a laboratory manual", CSH Press, 1988, Cold Spring Harbor
• Töpolt, Guenzi, Lubeseder-Martellato, Jörg, Naschberger, Stürzl: Proceedings of the 22nd Meeting of the European Society of Microcirculation (2002 ) ..
• Lubeseder-Martellato, Guenzi, Joerg, Topolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle, Matzen, Grimm, Ensoli and Stürzl: Am J Pathol 161 (5): 1749-59 (2002 ).
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Claims (14)

1. In vitro method for identification and/or quantification of guanylate binding protein-1 (GBP-1) or of fragments of this protein in the culture supernatant of a tissue sample, a sample of a body fluid or a sample of a cell culture supernatant, wherein the method comprises the following steps:

   (a) contacting the sample with a first receptor which specifically binds GBP-1, wherein the first receptor is an antibody, a Fab, Fv or scFv fragment of an antibody or an aptamer, and

   (b) detecting a specific binding of the receptor with GBP-1 or the fragment of this protein, wherein the fragment results from cleavage and is indicative of inflammatory diseases.
2. The method according to claim 1, furthermore comprising step (a') or (a") prior to contacting with the first receptor:

   (a') labelling the proteins contained in the sample; or

   (a") labelling the first receptor.
3. The method according to claim 1 or 2, wherein the receptor is immobilised on a surface prior to contacting with GBP-1 or fragments of this protein.
4. The method according to claim 1 or 2, wherein the receptor is immobilised on a surface after contacting with GBP-1 or fragments of this protein.
5. The method according to any one of claims 3 or 4, wherein the material of the surface is selected from a group consisting of Sepharose, latex, glass, polystyrene, polyvinyl, nitrocellulose and silicon.
6. The method according to any one of claims 3 to 5, wherein the surface is a membrane, a bead, a chip or a plate.
7. The method according to claim 6, furthermore comprising the step (a"') prior to the step of detecting a specific binding:

   (a"') precipitating the beads with the complexes which are bound thereto of the first receptor and GBP-1 or a fragment of this protein.
8. The method according to claim 7, wherein the detection of the specific binding in step (b) comprises a gel electrophoretic separation, optionally, furthermore, a Western blot analysis.
9. The method according to any one of claims 1 to 8, wherein for the detection of a specific binding of GBP-1 or a fragment of this protein with the first receptor in step (a), the sample is contacted with a second receptor for GBP-1, which binds to an epitope of GBP-1 or a fragment of this protein, which is accessible after the binding of the first receptor to GBP-1 or a fragment of this protein, and wherein the second receptor is an antibody, a Fab, Fv or scFv fragment of an antibody or an aptamer.
10. The method according to claim 9, wherein the second receptor for GBP-1 or fragments of this protein is labelled.
11. The method according to claim 10, wherein the label of the second receptor for GBP-1 or a fragment of this protein comprises a signalling system or is specifically recognised by a further, third receptor comprising a signalling system, wherein the third receptor is an antibody, a Fab, Fv or scFv fragment of an antibody or an aptamer.
12. The method according to claim 11, wherein the signalling system comprises an enzyme emitting the signal.
13. The method according to any one of claims 1 to 12, wherein the method is an ELISA, an EIA or an RIA.
14. The method according to any one of claims 1 to 13, wherein the method is carried out in an automated way.

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